This project concerns the cellular electrophysiology of cardiac muscle, with emphasis on the changes in membrane electrical properties that occur during development of chick and rat hearts and in cell culture. The parameters examined include K ion permeability, fast Na ion channels, slow Na ion and Ca ions channels, (Na,K)-ATPase, cyclic AMP levels, and ultrastructure. We are attempting to learn what factors control membrane differentiation in situ, e.g., whether neurotropic factors are involved. In answering these questions, young and old embryonic hearts will be placed in organ culture to determine what factors control membrane electrical differentiation in vitro. In addition, we will trypsin-disperse young and old embryonic hearts and prepare cell cultures (denervated) as monolayers and as spherical reaggregates. We will attempt to define what factors cause some cultured myocardial cells to revert back (partially dedifferentiate) towards the young embryonic state, whereas other cell cultures retain (or regain) their highly differentiated state. One approach to be used is the addition of RNA-enriched fractions obtained from adult hearts and of specific protein fractions prepared from adult hearts and from serum. Spherical reaggregate cultures will be characterized with respect to degree of electrical coupling and presence of pharmacological receptors. We will also continue to elucidate the properties of myocardial slow channels, including their ionic specificity, activation energy, etc. A search will be made for other agents which make more slow channels become available for voltage activation, and how this is brought about. We will test our hypothesis that phosphorylation of protein constituent of the slow channel, by a cyclic AMP-dependent protein kinase, makes the channel available for voltage activation, and that this is one of the mechanisms by which some positive inotropic agents and neurotransmitters act. We will test whether the peculiar property of myocardial slow channels, namely energy dependence, serves to protect the heart under adverse conditions of hypoxia or regional ischemia.